Mass spectrometry analysis of microorganisms in samples

ABSTRACT

The invention generally relates to systems and methods for mass spectrometry analysis of microorganisms in samples.

RELATED APPLICATIONS

The present application is a continuation of U.S. nonprovisionalapplication Ser. No. 14/151,220, filed Jan. 9, 2014, which is acontinuation of U.S. nonprovisional application Ser. No. 13/926,645,filed Jun. 25, 2013, which is a continuation-in-part of U.S.nonprovisional application Ser. No. 13/265,110, filed Jan. 31, 2012,which application is a national phase application and claims the benefitof and priority to PCT/US2010/032881, filed Apr. 29, 2010, which claimspriority to and the benefit of U.S. provisional patent application Ser.No. 61/174,215, filed Apr. 30, 2009, U.S. provisional patent applicationSer. No. 61/246,707 filed Sep. 29, 2009, and U.S. provisional patentapplication Ser. No. 61/308,332, filed Feb. 26, 2010, the content ofeach of which is incorporated by reference herein in its entirety.

TECHNICAL FIELD

The invention generally relates to systems and methods for massspectrometry analysis of microorganisms in samples.

BACKGROUND

Mass spectrometry is a very sensitive analytical method used forimportant research and for applications of analytical chemistry,particularly life science. Electrospray ionization (ESI) is generallyregarded as the best-characterized and most efficient method forionization of molecules in solution phase. The process can beconveniently divided into three stages: droplet formation, dropletevaporation and ion formation (Gaskell, S. J. Journal of MassSpectrometry 1997, 32, 677-688). When a strong electric field is appliedto a solution flowing through a mass spectrometer probe, a Taylor coneis formed at the tip of the probe, resulting in a mist of small dropletsbeing emitted from the tip of this cone. Due to the evaporation of thefree droplets and Coulombic forces, ions of sample analyte are produced.The ions enter a mass spectrometer and are subsequently analyzed.

A problem with ESI is that sample preparation is still a necessary stepbefore ESI can be used for analysis of many types of samples. Prior toanalyzing a sample by ESI mass spectrometry, the sample will undergoextraction and filtration protocols to purify the sample, for example toremove salts and detergents. Such protocols are complex, time-consuming,and expensive. Further, reagents used during the purification processcan interfere with subsequent analysis of a target analyte in thepurified sample. Additionally, samples that are not in solution must bedissolved as well as purified prior to ESI analysis.

More recently, the concept of ambient ionization has been developed, andnow this family of ambient ionization has more than twenty members, suchas desorption electrospray ionization (DESI) and direct analysis in realtime (DART). Ambient ionization by mass spectrometry allows theionization of analytes under an ambient environment from condensed-phasesamples without much or even any sample preparation and/orpre-separation, offering a solution for real time and in situ analysisfor complex mixtures and biological samples. These ambient ionizationmethods are leading are extending the mass spectrometry revolution inlife science, environment monitoring, forensic applications andtherapeutic analysis. However, the above described ambient ionizationtechniques still require pneumatic assistance, a continuous flow ofsolvent, and a high voltage power supply for the analysis of samples.

There is an unmet need for systems and methods that can combine samplepreparation and pre-treatment and the ionization process for massanalysis of samples that do not require pneumatic assistance or acontinuous flow of solvent for the analysis of the samples.

SUMMARY

The invention generally relates to new systems and methods of generatingions from fluids and solid samples for mass spectrometric analysis.Porous materials, such as filter paper or similar materials are used tohold and transfer liquids, and ions are generated directly from theedges of the materials when a high electric voltage is applied to thematerials. The porous material is kept discrete (i.e., separate ordisconnected from) from a flow of solvent. Instead, a sample is eitherspotted onto the porous material or the porous material is wetted andused to swab a surface containing the sample. The porous material withspotted or swabbed sample is then wetted and connected to a high voltagesource to produce ions of the sample which are subsequently analyzed.The sample is transported through the porous material without the needof a separate solvent flow.

Devices and methods of the invention combine sample preparation andpre-treatment with the ionization process needed for mass analysis ofsamples. Device and methods of the invention allow for rapid and directanalysis of chemicals in raw biological samples of complex matrices,such as biofluids and tissues, without sample preparation. In particularembodiments, devices and methods of the invention allow for the analysisof a dried spots of blood or urine.

An aspect of the invention provides a mass spectrometry probe includinga porous material connected to a high voltage source, in which theporous material is discrete from a flow of solvent. Exemplary porousmaterials include paper, e.g., filter paper, or PVDF membrane. Theporous material can be of any shape. In certain embodiments, the porousmaterial is provided as a triangular piece.

In certain embodiments, the probe further includes a discrete amount ofa solvent, e.g., a droplet or droplets, applied to the porous material.The solvent is applied as a droplet or droplets, and in an amountsufficient to wet the porous material. Once applied to the porousmaterial, the solvent can assist transport of the sample through theporous material. The solvent can contain an internal standard. Thesolvent/substrate combination can allow for differential retention ofsample components with different chemical properties. In certainembodiments, the solvent minimizes salt and matrix effects. In otherembodiments, the solvent includes chemical reagents that allow foron-line chemical derivatization of selected analytes.

Another aspect of the invention provides a system for analyzing a samplematerial including, a probe including a porous material connected to ahigh voltage source, in which the porous material is kept separate froma flow of solvent, and a mass analyzer. The mass analyzer can be that ofa benchtop mass spectrometer or a handheld mass spectrometer. Exemplarymass analyzers include a quadrupole ion trap, a rectilinear ion trap, acylindrical ion trap, a ion cyclotron resonance trap, and an orbitrap.

Another aspect of the invention includes a method for analyzing a sampleincluding, contacting a sample to a porous material, in which the porousmaterial is kept separate from a flow of solvent, applying a highvoltage to the porous material to generate ions of an analyte in thesample that are expelled from the porous material, and analyzing theexpelled ions. The method can further include applying a discreteamount, e.g., a droplet or droplets, of a solvent to the porousmaterial. In certain embodiments, analyzing involves providing a massanalyzer to generate a mass spectrum of analytes in the sample.

In certain embodiments, the sample is a liquid. In other embodiments,the sample is a solid. In embodiments in which the sample is a solid,the porous material can be used to swab the sample from a surface. Asolvent can be applied to the porous material prior to or after thesolid has been swabbed. Exemplary samples include chemical species orbiological species.

Another aspect of the invention provides a method of ionizing a sampleincluding applying a high voltage to a porous material to generate ionsof an analyte in the sample, in which the porous material remainsseparate from a solvent flow. Exemplary porous materials include paperor PVDF membrane.

BRIEF DESCRIPTION OF THE FIGURES

FIG. 1A is a drawing of a sample solution being fed to a piece of paperfor electrospray ionization. FIG. 1B is a drawing of a sample solutionpre-spotted onto the paper and a droplet of solvent being subsequentlysupplied to the paper for electrospray ionization.

FIG. 2A is a MS spectrum of heroin (concentration: 1 ppm, volume: 10 μl,solvent: MeOH/H₂O/HOAc (50:49:1, v/v/v)) using probes of the invention.FIG. 2B is a MS/MS spectrum of heroin (concentration: 1 ppb, volume: 10μl, solvent: MeOH/H₂O/HOAc (50:49:1, v/v/v)).

FIG. 3A is a MS spectrum of caffeine (concentration: 10 ppm, volume: 10μl, solvent: MeOH/H₂O/HOAc (50:49:1, v/v/v)) using probes of theinvention. FIG. 3B is a MS/MS spectrum of caffeine (concentration: 10ppb, volume: 10 μl, solvent: MeOH/H₂O/HOAc (50:49:1, v/v/v)).

FIG. 4A is a MS spectrum of benzoylecgonine (concentration: 10 ppm,volume: 10 μA solvent: MeOH/H₂O/HOAc (50:49:1, v/v/v)) using probes ofthe invention. FIG. 4B is a MS/MS spectrum of benzoylecgonine(concentration: 10 ppb, volume: 10 μl, solvent: MeOH/H₂O/HOAc (50:49:1,v/v/v)).

FIG. 5A is a MS spectrum of serine (concentration: 1 ppm, volume: 10 μl,solvent: MeOH/H₂O/HOAc (50:49:1, v/v/v)) using probes of the invention.FIG. 5B is a MS/MS spectrum of serine (concentration: 100 ppb, volume:10 μl, solvent: MeOH/H₂O/HOAc (50:49:1, v/v/v)).

FIG. 6A is a MS spectrum of peptide bradykinin2-9 (concentration: 10ppm, volume: 10 μl, solvent: MeOH/H₂O/HOAc (50:49:1, v/v/v)) usingprobes of the invention. FIG. 6B is a MS/MS spectrum of bradykinin2-9(concentration: 1 ppm, volume: 10 μl, solvent: MeOH/H₂O/HOAc (50:49:1,v/v/v)).

FIG. 7A is a MS/MS spectrum showing that heroin can be detected fromwhole blood sample by a “spot” method. FIG. 7B shows the MS/MS spectrumof the blood spot without heroin.

FIG. 8A MS/MS spectrum shows heroin can be detected from raw urinesample by a “spot” method. FIG. 8B shows the MS/MS spectrum of the urinespot without heroin.

FIG. 9A is a MS spectrum showing the caffeine detected from a cola drinkwithout sample preparation. FIG. 9B is a MS spectrum showing caffeinedetected from coffee powder. A paper slice was used to collect thecoffee powder from a coffee bag by swabbing the surface.

FIGS. 10A-B show MS spectra of urine analysis without samplepreparation. FIG. 10A is a MS spectrum showing that caffeine wasdetected in urine from a person who consumed coffee. FIG. 10B is a MSspectrum showing that caffeine was not detected in urine from a personwho had not consumed any coffee.

FIGS. 11A-B are MS spectra showing the difference between peptideanalysis (10 ppm of bradykinin 2-9) on (FIG. 11A) paper triangle and(FIG. 11B) PVDF membrane using the same parameters (˜2 kV, Solvent:MeOH:H₂O=1:1).

FIGS. 12A-D show direct MS spectra of plant tissues using sliced tissuesof four kinds of plants. (FIG. 12A) Onion, (FIG. 12B) Spring onion, andtwo different leaves (FIG. 12C) and (FIG. 12D).

FIGS. 13A-B show MS/MS spectra of Vitamin C. FIG. 13A direct analysis ofonion without sample preparation. FIG. 13B using standard solution.

FIG. 14A is a picture showing dried blood spot analysis on paper; 0.4 μLof whole blood is applied directly to a triangular section ofchromatography paper (typically height 10 mm, base 5 mm). A copper clipholds the paper section in front of the inlet of an LTQ massspectrometer (Thermo Fisher Scientific, San Jose, Calif.) and a DCvoltage (4.5 kV) is applied to the paper wetted with 10 μLmethanol/water (1:1 v/v). FIG. 14B shows the molecular structure ofimatinib (GLEEVEC) and paper spray tandem mass spectrum of 0.4 μL wholeblood containing 4 μg/mL imatinib. Imatinib is identified and quantified(inset) by the MS/MS transition m/z 494→m/z 394 (inset). FIG. 14C showsa quantitative analysis of whole blood spiked with imatinib (62.5-4μg/mL) and its isotopomers imatinib-d8 (1 μg/mL). Inset plot shows lowconcentration range.

FIG. 15 is a paper spray mass spectrum of angiotensin I solution. Theinset shows an expanded view over the mass range 630-700.

FIG. 16 is a mass spectrum showing direct analysis of hormones in animaltissue by probes of the invention.

FIGS. 17A-B are mass spectra showing direct analysis of human prostatetumor tissue and normal tissue.

FIG. 18 is a mass spectrum of whole blood spiked with 10 μg/mL atenolol.The data was obtained by combining systems and methods of the inventionwith a handheld mass spectrometer.

FIGS. 19A-F show mass spectra of cocaine sprayed from six differenttypes of paper (Whatman filter paper with different pore sizes: (FIG.19A) 3 μm, (FIG. 19B) 4-7 μm, (FIG. 19C) 8 μm, and (FIG. 19D) 11 μm,(FIG. 19E) glass fiber paper and (FIG. 19F) chromatography paper). Thespray voltage was 4.5 kV.

FIG. 20A shows a schematic setup for characterizing the spatialdistribution of paper spray. FIG. 20B is a 2D contour plot showing therelative intensity of m/z 304 when the probe is moved in the x-y planewith respect to the inlet of the mass spectrometer. FIG. 20C is a graphshowing signal duration of m/z 304 when loading cocaine solution onpaper with different concentrations or volumes, or sealed by Teflonmembrane.

FIGS. 21A-D are a set of MS spectra of pure chemical solutions and theircorresponding MS/MS spectra. Spectra were obtained for (FIG. 21A)serine, (FIG. 21B) methadone, (FIG. 21C) roxithromycin, and (FIG. 21D)bradykinin 2-9.

FIGS. 22A-G are a set of mass spectra showing analysis of chemicals fromcomplex mixtures and direct analysis from surfaces without samplepreparation. FIGS. 22A-B are mass spectra of COCA-COLA (cola drink),which was directly analyzed on paper in both of (FIG. 22A) positive and(FIG. 22B) negative mode. FIG. 22C is a mass spectrum of caffeine. FIG.22D is a mass spectrum of potassium benzoate. FIG. 22E is a massspectrum of acesulfame potassium. FIG. 22F is a mass spectrum ofcaffeine detected from urine. FIG. 22G is a mass spectrum of heroindetected directly from a desktop surface after swabbing of the surfaceby probes of then invention.

FIG. 23A shows images of a probe of the invention used for bloodanalysis. In this embodiment, the porous material is paper. The panel onthe left is prior to spotting with whole blood. The panel in the middleis after spotting with whole blood and allowing the spot to dry. Thepanel on the right is after methanol was added to the paper and allowedto travel through the paper. The panel on the right shows that themethanol interacts with the blood spot, causing analytes to travel tothe tip of the paper for ionization and analysis. FIG. 23B is a massspectrum of Atenolol from whole blood FIG. 23C is a mass spectrum ofheroin from whole blood.

FIGS. 24A-C show analysis of two dyes, methylene blue (m/z 284) andmethyl violet (m/z 358.5), separated by TLC. Dye mixture solution (0.1μl of a 1 mg/mL solution) was applied onto the chromatography paper (4cm×0.5 cm) and dried before TLC and paper spray MS analysis.

FIGS. 25A-E show different shapes, thicknesses, and angles for probes ofthe invention. FIG. 25A shows sharpness. FIG. 25B shows angle of thetip. FIG. 25C shows thickness of the paper. FIG. 25D shows a device withmultiple spray tips. FIG. 25E shows a DBS card with micro spray tipsfabricated with sharp needles.

FIGS. 26A-B are a set of mass spectra of imatinib from human serum usingdirect spray from a C4 zip-tip of conical shape. Human serum samples(1.5 μL each) containing imatinib were passed through the porous C4extraction material three times and then 3 μL methanol was added ontothe zip-tip with 4 kV positive DC voltage applied to produce the spray.FIG. 26A shows a MS spectrum for 5 μg/mL. FIG. 26B shows a MS/MSspectrum for 5 ng/mL.

FIG. 27A is a picture showing different tip angles for probes of theinvention. From left to right, the angles are 30, 45, 90, 112, 126degree, respectively. FIG. 27B is a graph showing the effect of angle onMS signal intensity. All MS signals were normalized to the MS signalusing the 90 degree tip.

FIG. 28A is a picture of a high-throughput probe device of theinvention. FIG. 28B shows spray from a single tip of the device into aninlet of a mass spectrometer. FIG. 28C is a set of mass spectra showingMS signal intensity in high-throughput mode.

FIG. 29A is a schematic depicting a protocol for direct analysis ofanimal tissue using probes of the invention. FIGS. 29B-D are massspectra showing different chemicals detected in the tissue.

FIG. 30A shows a mass spectral analysis of a dried serum spot on plainpaper. FIG. 30B shows a mass spectrum analysis of a dried serum sport onpaper preloaded with betaine aldehyde (BA) chloride. FIG. 30C shows aMS/MS analysis of reaction product [M+BA]⁺ (m/z 488.6).

FIGS. 31A-B show MS/MS spectra recorded with modified (FIG. 31A) andunmodified (FIG. 31B) paper substrates.

FIG. 32 is a mass spectrum showing that ions can be generated using anegative ion source potential but positively charged ions aremass-analyzed.

FIG. 33A is a schematic showing the design of a sample cartridge withvolume control and overflowing vials. A soluble plug with internalstandard chemical is used to block the bottom of the volume controlvial. FIG. 33B shows a step-by-step process of applying blood samplesonto the cartridge to prepare a dried blood spot on paper from acontrolled volume of blood.

FIGS. 34A-B show mass spectra of agrochemicals that are present on alemon peel purchased from a grocery store and swabbed with paper.

FIG. 35 shows a design of a substrate for paper spray with multiplecorners. The angle of the corner to be used for spray is smaller thanthat of other corners.

FIGS. 36A-B show a spray tip fabricated on a piece of chromatographypaper using SU-8 2010 photoresist. FIG. 36C shows a MS spectrum ofmethanol/water solution containing a mixture of asparagines.

FIG. 37 shows an exemplary method of collecting microorganisms ontoprobes of the invention when the sample is a gas/aerosol.

FIG. 38 shows an exemplary method of collecting microorganisms ontoprobes of the invention when the sample is a liquid.

FIGS. 39A-B show mass spectra of E. coli. FIG. 39A is negative ion modeand FIG. 39B is positive ion mode.

FIG. 40 show a mass spectra of different microorganisms. The top panelis a mass spectrum of staphylococcus capitis. The bottom panel is a massspectrum of staphylococcus saprophyticus

FIG. 41 shows a mass spectrum of E. coli acquired in negative mode.

FIG. 42 shows a mass spectrum of E. coli acquired in positive mode.

FIG. 43 is a graph showing a principal component analysis of differentmicroorganisms.

FIG. 44 is a similarity comparison of different organisms.

FIGS. 45A-F show a workflow for comparing a mass spectrum of an unknownmicroorganism to a database including mass spectra of knownmicroorganisms to identify the unknown microorganism.

DETAILED DESCRIPTION

A new method of generating ions from fluids and solids for massspectrometry analysis is described. Porous materials, such as paper(e.g. filter paper or chromatographic paper) or other similar materialsare used to hold and transfer liquids and solids, and ions are generateddirectly from the edges of the material when a high electric voltage isapplied to the material (FIG. 1). The porous material is kept discrete(i.e., separate or disconnected) from a flow of solvent, such as acontinuous flow of solvent. Instead, sample is either spotted onto theporous material or swabbed onto it from a surface including the sample.The spotted or swabbed sample is then connected to a high voltage sourceto produce ions of the sample which are subsequently mass analyzed. Thesample is transported through the porous material without the need of aseparate solvent flow. Pneumatic assistance is not required to transportthe analyte; rather, a voltage is simply applied to the porous materialthat is held in front of a mass spectrometer.

In certain embodiments, the porous material is any cellulose-basedmaterial. In other embodiments, the porous material is a non-metallicporous material, such as cotton, linen wool, synthetic textiles, orplant tissue. In still other embodiments, the porous material is paper.Advantages of paper include: cost (paper is inexpensive); it is fullycommercialized and its physical and chemical properties can be adjusted;it can filter particulates (cells and dusts) from liquid samples; it iseasily shaped (e.g., easy to cut, tear, or fold); liquids flow in itunder capillary action (e.g., without external pumping and/or a powersupply); and it is disposable.

In certain embodiments, the porous material is integrated with a solidtip having a macroscopic angle that is optimized for spray. In theseembodiments, the porous material is used for filtration,pre-concentration, and wicking of the solvent containing the analytesfor spray at the solid type.

In particular embodiments, the porous material is filter paper.Exemplary filter papers include cellulose filter paper, ashless filterpaper, nitrocellulose paper, glass microfiber filter paper, andpolyethylene paper. Filter paper having any pore size may be used.Exemplary pore sizes include Grade 1 (11 μm), Grade 2 (8 μm), Grade 595(4-7 μm), and Grade 6 (3 μm), Pore size will not only influence thetransport of liquid inside the spray materials, but could also affectthe formation of the Taylor cone at the tip. The optimum pore size willgenerate a stable Taylor cone and reduce liquid evaporation. The poresize of the filter paper is also an important parameter in filtration,i.e., the paper acts as an online pretreatment device. Commerciallyavailable ultra filtration membranes of regenerated cellulose, with poresizes in the low nm range, are designed to retain particles as small as1000 Da. Ultra filtration membranes can be commercially obtained withmolecular weight cutoffs ranging from 1000 Da to 100,000 Da.

Probes of the invention work well for the generation of micron scaledroplets simply based on using the high electric field generated at anedge of the porous material. In particular embodiments, the porousmaterial is shaped to have a macroscopically sharp point, such as apoint of a triangle, for ion generation. Probes of the invention mayhave different tip widths. In certain embodiments, the probe tip widthis at least about 5 μm or wider, at least about 10 μm or wider, at leastabout 50 μm or wider, at least about 150 μm or wider, at least about 250μm or wider, at least about 350 μm or wider, at least about 400 μm orwider, at least about 450 μm or wider, etc. In particular embodiments,the tip width is at least 350 μm or wider. In other embodiments, theprobe tip width is about 400 μm. In other embodiments, probes of theinvention have a three dimensional shape, such as a conical shape.

As mentioned above, no pneumatic assistance is required to transport thedroplets. Ambient ionization of analytes is realized on the basis ofthese charged droplets, offering a simple and convenient approach formass analysis of solution-phase samples.

Sample solution is directly applied on the porous material held in frontof an inlet of a mass spectrometer without any pretreatment. Then theambient ionization is performed by applying a high potential on thewetted porous material. In certain embodiments, the porous material ispaper, which is a type of porous material that contains numerical poresand microchannels for liquid transport. The pores and microchannels alsoallow the paper to act as a filter device, which is beneficial foranalyzing physically dirty or contaminated samples.

In other embodiments, the porous material is treated to producemicrochannels in the porous material or to enhance the properties of thematerial for use as a probe of the invention. For example, paper mayundergo a patterned silanization process to produce microchannels orstructures on the paper. Such processes involve, for example, exposingthe surface of the paper totridecafluoro-1,1,2,2-tetrahydrooctyl-1-trichlorosilane to result insilanization of the paper. In other embodiments, a soft lithographyprocess is used to produce microchannels in the porous material or toenhance the properties of the material for use as a probe of theinvention. In other embodiments, hydrophobic trapping regions arecreated in the paper to pre-concentrate less hydrophilic compounds.

Hydrophobic regions may be patterned onto paper by usingphotolithography, printing methods or plasma treatment to definehydrophilic channels with lateral features of 200-1000 μm. See Martinezet al. (Angew. Chem. Int. Ed. 2007, 46, 1318-1320); Martinez et al.(Proc. Natl. Acad. Sci. USA 2008, 105, 19606-19611); Abe et al. (Anal.Chem. 2008, 80, 6928-6934); Bruzewicz et al. (Anal. Chem. 2008, 80,3387-3392); Martinez et al. (Lab Chip 2008, 8, 2146-2150); and Li et al.(Anal. Chem. 2008, 80, 9131-9134), the content of each of which isincorporated by reference herein in its entirety. Liquid samples loadedonto such a paper-based device can travel along the hydrophilic channelsdriven by capillary action.

Another application of the modified surface is to separate orconcentrate compounds according to their different affinities with thesurface and with the solution. Some compounds are preferably absorbed onthe surface while other chemicals in the matrix prefer to stay withinthe aqueous phase. Through washing, sample matrix can be removed whilecompounds of interest remain on the surface. The compounds of interestcan be removed from the surface at a later point in time by otherhigh-affinity solvents. Repeating the process helps desalt and alsoconcentrate the original sample.

Methods and systems of the invention use a porous material, e.g., paper,to hold and transport analytes for mass spectral analysis. Analytes insamples are pre-concentrated, enriched and purified in the porousmaterial in an integrated fashion for generation of ions withapplication of a high voltage to the porous material. In certainembodiments, a discrete amount of transport solution (e.g., a droplet ora few droplets) is applied to assist movement of the analytes throughthe porous material. In certain embodiments, the analyte is already in asolution that is applied to the porous material. In such embodiments, noadditional solvent need be added to the porous material. In otherembodiments, the analyte is in a powdered sample that can be easilycollected by swabbing a surface. Systems and methods of the inventionallow for analysis of plant or animal tissues, or tissues in livingorganisms.

Methods and systems of the invention can be used for analysis of a widevariety of small molecules, including epinephrine, serine, atrazine,methadone, roxithromycin, cocaine and angiotensin I. All display highquality mass and MS/MS product ion spectra (see Examples below) from avariety of porous surfaces. Methods and systems of the invention allowfor use of small volumes of solution, typically a few μL, with analyteconcentrations on the order of 0.1 to 10 μg/mL (total amount analyte 50pg to 5 ng) and give signals that last from one to several minutes.

Methods and systems of the invention can be used also for analysis of awide variety of biomolecules, including proteins and peptides. Methodsof the invention can also be used to analyze oligonucleotides from gels.After electrophoretic separation of oligonucleotides in the gel, theband or bands of interest are blotted with porous material using methodsknown in the art. The blotting results in transfer of at least some ofthe oligonucleotides in the band in the gel to the porous material. Theporous material is then connected to a high voltage source and theoligonucleotides are ionized and sprayed into a mass spectrometer formass spectral analysis.

Methods and systems of the invention can be used for analysis of complexmixtures, such as whole blood or urine. The typical procedure for theanalysis of pharmaceuticals or other compounds in blood is a multistepprocess designed to remove as many interferences as possible prior toanalysis. First, the blood cells are separated from the liquid portionof blood via centrifugation at approximately 1000×g for 15 minutes(Mustard, J. F.; Kinlough-Rathbone, R. L.; Packham, M. A. Methods inEnzymology; Academic Press, 1989). Next, the internal standard is spikedinto the resulting plasma and a liquid-liquid or solid-phase extractionis performed with the purpose of removing as many matrix chemicals aspossible while recovering nearly all of the analyte (Buhrman, D. L.;Price, P. I.; Rudewicz, P. J. Journal of the American Society for MassSpectrometry 1996, 7, 1099-1105). The extracted phase is typically driedby evaporating the solvent and then resuspended in the a solvent used asthe high performance liquid chromatography (HPLC) mobile phase(Matuszewski, B. K.; Constanzer, M. L.; Chavez-Eng, C. M., Ithaca, N.Y.,Jul. 23-25 1997; 882-889). Finally, the sample is separated in thecourse of an HPLC run for approximately 5-10 minutes, and the eluent isanalyzed by electrospray ionization-tandem mass spectrometry(Hopfgartner, G.; Bourgogne, E. Mass Spectrometry Reviews 2003, 22,195-214).

Methods and systems of the invention avoid the above sample work-upsteps. Methods and systems of the invention analyze a dried blood spotsin a similar fashion, with a slight modification to the extractionprocedure. First, a specialized device is used to punch out identicallysized discs from each dried blood spot. The material on these discs isthen extracted in an organic solvent containing the internal standard(Chace, D. H.; Kalas, T. A.; Naylor, E. W. Clinical Chemistry 2003, 49,1797-1817). The extracted sample is dried on the paper substrate, andthe analysis proceeds as described herein.

Examples below show that methods and systems of the invention candirectly detect individual components of complex mixtures, such ascaffeine in urine, 50 pg of cocaine on a human finger, 100 pg of heroinon a desktop surface, and hormones and phospholipids in intact adrenaltissue, without the need for sample preparation prior to analysis (SeeExamples below). Methods and systems of the invention allow for simpleimaging experiments to be performed by examining, in rapid succession,needle biopsy tissue sections transferred directly to paper.

Analytes from a solution are applied to the porous material forexamination and the solvent component of the solution can serve as theelectrospray solvent. In certain embodiments, analytes (e.g., solid orsolution) are pre-spotted onto the porous material, e.g., paper, and asolvent is applied to the material to dissolve and transport the analyteinto a spray for mass spectral analysis.

In certain embodiments, a solvent is applied to the porous material toassist in separation/extraction and ionization. Any solvents may be usedthat are compatible with mass spectrometry analysis. In particularembodiments, favorable solvents will be those that are also used forelectrospray ionization. Exemplary solvents include combinations ofwater, methanol, acetonitrile, and THF. The organic content (proportionof methanol, acetonitrile, etc. to water), the pH, and volatile salt(e.g. ammonium acetate) may be varied depending on the sample to beanalyzed. For example, basic molecules like the drug imatinib areextracted and ionized more efficiently at a lower pH. Molecules withoutan ionizable group but with a number of carbonyl groups, like sirolimus,ionize better with an ammonium salt in the solvent due to adductformation.

In certain embodiments, a multi-dimensional approach is undertaken. Forexample, the sample is separated along one dimension, followed byionization in another dimension. In these embodiments, separation andionization can be individually optimized, and different solvents can beused for each phase.

In other embodiments, transporting the analytes on the paper isaccomplished by a solvent in combination with an electric field. When ahigh electric potential is applied, the direction of the movement of theanalytes on paper is found to be related to the polarity of theircharged forms in solution. Pre-concentration of the analyte before thespray can also be achieved on paper by placing an electrode at a pointon the wetted paper. By placing a ground electrode near the paper tip, astrong electric field is produced through the wetted porous materialwhen a DC voltage is applied, and charged analytes are driven forwardunder this electric field. Particular analytes may also be concentratedat certain parts of the paper before the spray is initiated.

In certain embodiments, chemicals are applied to the porous material tomodify the chemical properties of the porous material. For example,chemicals can be applied that allow differential retention of samplecomponents with different chemical properties. Additionally, chemicalscan be applied that minimize salt and matrix effects. In otherembodiments, acidic or basic compounds are added to the porous materialto adjust the pH of the sample upon spotting. Adjusting the pH may beparticularly useful for improved analysis of biological fluids, such asblood. Additionally, chemicals can be applied that allow for on-linechemical derivatization of selected analytes, for example to convert anon-polar compound to a salt for efficient electrospray ionization.

In certain embodiments, the chemical applied to modify the porousmaterial is an internal standard. The internal standard can beincorporated into the material and released at known rates duringsolvent flow in order to provide an internal standard for quantitativeanalysis. In other embodiments, the porous material is modified with achemical that allows for pre-separation and pre-concentration ofanalytes of interest prior to mass spectrum analysis.

The spray droplets can be visualized under strong illumination in thepositive ion mode and are comparable in size to the droplets emittedfrom a nano-electrospray ion sources (nESI). In the negative ion mode,electrons are emitted and can be captured using vapor phase electroncapture agents like benzoquinone. Without being limited by anyparticular theory or mechanism of action, it is believed that the highelectric field at a tip of the porous material, not the fields in theindividual fluid channels, is responsible for ionization.

The methodology described here has desirable features for clinicalapplications, including neotal screening, therapeutic drug monitoringand tissue biopsy analysis. The procedures are simple and rapid. Theporous material serves a secondary role as a filter, e.g., retainingblood cells during analysis of whole blood. Significantly, samples canbe stored on the porous material and then analyzed directly from thestored porous material at a later date without the need transfer fromthe porous material before analysis. Systems of the invention allow forlaboratory experiments to be performed in an open laboratoryenvironment.

INCORPORATION BY REFERENCE

References and citations to other documents, such as patents, patentapplications, patent publications, journals, books, papers, webcontents, have been made throughout this disclosure. All such documentsare hereby incorporated herein by reference in their entirety for allpurposes.

EQUIVALENTS

Various modifications of the invention and many further embodimentsthereof, in addition to those shown and described herein, will becomeapparent to those skilled in the art from the full contents of thisdocument, including references to the scientific and patent literaturecited herein. The subject matter herein contains important information,exemplification and guidance that can be adapted to the practice of thisinvention in its various embodiments and equivalents thereof.

EXAMPLES

The following examples are intended to further illustrate certainembodiments of the invention, and are not to be construed to limit thescope of the invention. Examples herein show that mass spectrometryprobes of the invention can ionize chemical and biological samples,allowing for subsequent mass analysis and detection. An exemplary probewas constructed as a paper triangle, which was used to generate micronscale droplets by applying a high potential on the paper. The analyteswere ionized from these electrically charged droplets and transportedinto a conventional mass spectrometer.

Examples below show that a wide range of samples could be directlyanalyzed in the ambient environment by probes of the invention in bothof pure state and complex mixtures. The results showed that paper-basedspray has the following benefits: it operated without sheath gas, i.e.,few accessories were required for in situ analysis; biological samples(dried blood, urine) could be stored on the precut filter papers formonths before analysis; filter paper minimized matrix effects seen withelectrospray or nano electrospray in many samples (blood cells, salt andproteins) and enhanced the MS signal of chemicals in complex samples;powdered samples were easily collected by swabbing surfaces using paperpieces and then directly analyzed; the paper could be pretreated tocontain internal standards that were released at known rates duringsolvent flow in quantitative analysis; and the paper could be pretreatedto contain matrix suppression or absorption sites or to perform ionexchange or to allow on-line chemical derivatization of selectedanalytes.

Detection of most analytes was achieved as low as ppb levels (whenexamined as solutions) or in the low ng to pg range (when solids wereexamined) and the detection time was less than one minute. CertainExamples below provide a protocol for analyzing a dried blood spot,which can also be used for in situ analysis of whole blood samples. Thedried blood spot method is also demonstrated to be compatible with thestorage and transport of blood sample for blood screening and otherclinical tests.

Devices of the invention integrated the capabilities of sampling,pre-separation, pre-concentration and ionization. Methods and systems ofthe invention simplify the problem of sample introduction in massanalyzers.

Example 1 Construction of an MS Probe

Filter paper was cut into triangular pieces with dimensions of 10 mmlong and 5 mm wide and used as a sprayer (FIGS. 1A-B). A copper clip wasattached to the paper, and the paper was oriented to face an inlet of amass spectrometer (FIGS. 1A-B). The copper clip was mounted on a 3Dmoving stage to accurately adjust its position. A high voltage wasapplied to the copper clip and controlled by a mass spectrometer togenerate analyte ions for mass detection.

Samples were directly applied to the paper surface that served as asample purification and pre-concentration device. Filter paper allowedliquid samples to move through the hydrophilic network driven bycapillary action and electric effects and to transport them to the tipof the paper. Separation could take place during this transport process.Sample solution was sprayed from the tip and resulted in ionization andMS detection when a high voltage (˜4.5 kV) was applied to the papersurface.

All experiments were carried out with a Finnigan LTQ mass spectrometer(Thermo Electron, San Jose, Calif.). The typical temperature of thecapillary inlet was set at 150° C. while 30° C. for heroin detection.The lens voltage was set at 65 V for sample analysis and 240 V forsurvival yield experiment. Tandem mass spectra were collected usingcollision-induced dissociation (CID) to identify analytes in testedsamples, especially for complex mixtures and blood samples.

Example 2 Spray Generation

Spray was produced by applying a high potential on the wetted papertriangle. One paper triangle was placed in front of the inlet of LTQwith its sharp tip facing to the inlet, separated by 3 mm or more.Typically, 10 uL sample solution was applied to wet the paper triangle.The solution can wet or saturate the paper or form a thin layer ofliquid film on the surface of the paper. A high potential (3-5 kV) wasapplied between the paper triangle and mass inlet to generate anelectric field, which induced a charge accumulation on the liquid at thetip of paper triangle. The increasing coulombic force breaks the liquidto form charged droplets and then the solvent evaporated during theflight of droplets from the paper tip to the mass analyzer. Paper sprayrequired no sheath gas, heating or any other assistance to remove thesolvent.

When liquid accumulated on the paper triangle, a Taylor cone wasobserved at the tip when examined with a microscope. The droplets formedwere clearly visible under strong illumination. The Taylor cone andvisible spray disappeared after a short time of evaporation and spray.However, the mass signal lasted for a much longer period (severalminutes). This revealed that the paper triangle could work in two modesfor mass analysis. In a first mode, the liquid was transported insidethe paper at a rate faster than the liquid could be consumed as spray atthe paper tip, resulting in a large cone being formed at the paper tipand droplets being generated. In a second mode, the liquid transportinside the paper was not able to move at a rate fast enough to keep upwith the spray consumption, and droplets were not visible. However, itwas observed that ionization of analytes did take place. The first modeprovided ESI like mass spectra and the second mode provided spectra withsome of the features APCI spectra. In the latter case, the papertriangle played a role analogous to a conductive needle to generate ahigh electric field to ionize the molecules in the atmosphere. It wasobserved that the mass signal in the first mode was stronger than themass signal in the second mode by approximately two orders of magnitudeunder the conditions and for the samples tested.

Example 3 Probe Considerations

Probe Materials

A number of porous materials were tested to generate charged dropletsfor mass spectrometry. The materials were shaped into triangles havingsharp tips and sample solution was then applied to the constructedprobes. Data herein show that any hydrophilic and porous substrate couldbe used successfully, including cotton swab, textile, plant tissues aswell as different papers. The porous network or microchannels of thesematerials offered enough space to hold liquid and the hydrophilicenvironment made it possible for liquid transport by capillary action.Hydrophobic and porous substrates could also be used successfully withproperly selected hydrophobic solvents.

For further investigation, six kinds of commercialized papers wereselected and qualitatively tested to evaluate their capabilities inanalyte detection. Filter papers and chromatography paper were made fromcellulose, while glass microfiber filter paper was made from glassmicrofiber. FIGS. 19A-F shows the mass spectra of cocaine detection onthose papers. The spectrum of glass fiber paper (FIG. 19E) was uniquebecause the intensity of background was two orders of magnitude lowerthan other papers and the cocaine peak (m/z, 304) could not beidentified.

It was hypothesized that the glass fiber paper was working on mode IIand prohibiting efficient droplet generation, due to the relative largethickness (˜2 mm). This hypothesis was proved by using a thin layerpeeled from glass fiber paper for cocaine detection. In that case, theintensity of the background increased and a cocaine peak was observed.All filter papers worked well for cocaine detection, (FIGS. 19A-D).Chromatography paper showed the cleanest spectrum and relative highintensity of cocaine (FIG. 19F).

Probe Shape and Tip Angle

Many different probe shapes were investigated with respect to generatingdroplets. A preferred shape of the porous material included at least onetip. It was observed that the tip allowed ready formation of a Taylorcone. A probe shape of a triangle was used most often. As shown in FIGS.25A-C, the sharpness of the tip, the angle of the tip (FIGS. 27A-B), andthe thickness of the paper substrate could effect the spraycharacteristics. The device of a tube shape with multiple tips (FIG.25D) is expected to act as a multiple-tip sprayer, which should haveimproved spray efficiency. An array of micro sprayers can also befabricated on a DBS card using sharp needles to puncture the surface(FIG. 25E).

Example 4 Configuration of Probe with Inlet of a Mass Spectrometer

A paper triangle was mounted on a 2D moving stage to determine how themass signal was affected by the relative positions of the paper triangleand the mass spectrometer inlet. The paper triangle was moved 8 cm inthe y-direction in a continuous manner and 3 cm in the x-direction witha 2 mm increment for each step (FIG. 20A). Cocaine solution (1 ug/mL,methanol/water, 1:1 v/v) was continuously fed onto the paper surface.The mass spectrum was continuously recorded during the entire scan. Acontour plot of the peak intensity of protonated cocaine (m/z, 304) wascreated from the normalized data extracted from the mass spectrum (FIG.20B). The contour plot shows that it was not necessary for the papertriangle to be placed directly in-line with the inlet of the massspectrometer to generate droplets.

Spray duration was also tested (FIG. 20C). Paper triangles (size 10 mm,5 mm) were prepared. First, 10 uL solutions were applied on the papertriangles with different concentration of 0.1, 1 and 10 ug/mL. The spraytime for each paper was just slightly varied by the difference ofconcentration. After that, 1 ug/mL cocaine solutions were applied on thepaper triangles with different volumes of 5 uL, 10 uL and 15 uL. Thespray times showed a linear response followed by the increasing samplevolumes.

In another test, the paper was sealed with a PTFE membrane to preventevaporation of solution, which prolonged the spray time by about threetimes. These results indicate that paper spray offers long enough timeof spray for data acquisition even using 5 uL solution, and theintensity of signal is stable during the entire spray period.

Example 5 Separation and Detection

Probes of the invention include a porous material, such as paper, thatcan function to both separate chemicals in biological fluids before insitu ionization by mass spectrometry. In this Example, the porousmaterial for the probe was chromatography paper. As shown in FIG. 24A-C,a mixture of two dyes was applied to the paper as a single spot. Thedyes were first separated on the paper by TLC (thin layer chromatograph)and the separated dyes were examined using MS analysis by methods of theinvention with the paper pieces cut from the paper media (FIGS. 24A-C).Data show the separate dyes were detected by MS analysis (FIGS. 24A-C).

The chromatography paper thus allowed for sample collection, analyteseparation and analyte ionization. This represents a significantsimplification of coupling chromatography with MS analysis.Chromatography paper is a good material for probes of the inventionbecause such material has the advantage that solvent movement is drivenby capillary action and there is no need for a syringe pump. Anotheradvantage is that clogging, a serious problem for conventionalnanoelectrospray sources, is unlikely due to its multi-porouscharacteristics. Therefore, chromatography paper, a multi-porousmaterial, can be used as a microporous electrospray ionization source.

Example 6 Pure Compounds: Organic Drugs, Amino Acids, and Peptides

As already described, probes and methods of the invention offer a simpleand convenient ionization method for mass spectrometry. Paper triangleswere spotted with different compounds and connected to a high voltagesource to produce ions. All experiments were carried out with a FinniganLTQ mass spectrometer (Thermo Electron, San Jose, Calif.). Data hereinshow that a variety of chemicals could be ionized in solution phase,including amino acid, therapeutic drugs, illegal drugs and peptides.

FIG. 2A shows an MS spectrum of heroin (concentration: 1 ppm, volume: 10μl, solvent: MeOH/H₂O/HOAc (50:49:1, v/v/v)) using probes of theinvention. FIG. 2B shows MS/MS spectrum of heroin (concentration: 1 ppb,volume: 10 μl, solvent: MeOH/H₂O/HOAc (50:49:1, v/v/v)).

FIG. 3A shows MS spectrum of caffeine (concentration: 10 ppm, volume: 10μl, solvent: MeOH/H₂O/HOAc (50:49:1, v/v/v)) using probes of theinvention. FIG. 3B shows MS/MS spectrum of caffeine (concentration: 10ppb, volume: 10 μl, solvent: MeOH/H₂O/HOAc (50:49:1, v/v/v)). Peak 167also exists in the blank spectrum with solvent and without caffeine.

FIG. 4A shows MS spectrum of benzoylecgonine (concentration: 10 ppm,volume: 10 μl, solvent: MeOH/H₂O/HOAc (50:49:1, v/v/v)) using probes ofthe invention. FIG. 4B shows MS/MS spectrum of benzoylecgonine(concentration: 10 ppb, volume: 10 μl, solvent: MeOH/H₂O/HOAc (50:49:1,v/v/v)).

FIG. 5A shows MS spectrum of serine (concentration: 1 ppm, volume: 10μl, solvent: MeOH/H₂O/HOAc (50:49:1, v/v/v)) using probes of theinvention. FIG. 5B shows MS/MS spectrum of serine (concentration: 100ppb, volume: 10 μl, solvent: MeOH/H₂O/HOAc (50:49:1, v/v/v)). Peak 74and 83 also exist in the blank spectrum with solvent and without serine.FIG. 21A shows MS spectrum of serine (m/z, 106) using probes of theinvention. FIG. 21A also shows MS/MS spectrum of serine (m/z, 106).

FIG. 21B shows MS spectrum of methadone (m/z, 310) using probes of theinvention. FIG. 21B also shows MS/MS spectrum of methadone (m/z, 310).FIG. 21C shows MS spectrum of roxithromycin (m/z, 837) using probes ofthe invention. FIG. 21B also shows MS/MS spectrum of roxithromycin (m/z,837).

FIG. 6A shows MS spectrum of peptide bradykinin2-9 (concentration: 10ppm, volume: 10 μl, solvent: MeOH/H₂O/HOAc (50:49:1, v/v/v)) usingprobes of the invention. FIG. 6B shows MS/MS spectrum of bradykinin2-9(concentration: 1 ppm, volume: 10 μl, solvent: MeOH/H₂O/HOAc (50:49:1,v/v/v)). The hump in the spectrum is assumed to be caused by polymers,such as polyethylene glycol (PEG), which are frequently added tomaterials in industry. FIG. 21D shows MS spectrum of bradykinin 2-9(m/z, 453) using probes of the invention. FIG. 21D also shows MS/MSspectrum of bradykinin 2-9 (m/z, 453). FIG. 21D further shows adductions [M+H] (m/z, 904), [M+2H]²⁺ (m/z, 453), [M+H+Na]²⁺ (m/z, 464) and[M+2Na]²⁺ (m/z, 475). The m/z 453 peak was double charged adduct ionconfirmed by the MS/MS spectrum.

FIGS. 11A-B are MS spectra showing the difference between peptideanalysis (10 ppm of bradykinin 2-9) on (FIG. 11A) paper slice and (FIG.11B) PVDF membrane using the same parameters (˜2 kV, Solvent:MeOH:H₂O=1:1).

Data herein show that probes of the invention work well over themass/charge range from 50 to over 1000 for detection of pure compounds.Data further shows that detection was achieved down to as low as 1 ng/mLfor most chemicals, including illegal drugs, such as heroin, cocaine andmethadone.

Example 7 Complex Mixtures

Complex mixtures such as urine, blood, and cola drink were examinedusing methods, devices, and systems of the invention. All experimentswere carried out with a Finnigan LTQ mass spectrometer (Thermo Electron,San Jose, Calif.).

FIG. 7A shows an MS/MS spectrum that shows that heroin was detected fromwhole blood sample by a “spot” method. 0.4 μl of whole blood samplecontaining 200 ppb heroin was applied on the center of the trianglepaper to form a 1 mm² blood spot. After the spot was dry, 10 μl ofsolvent (MeOH/H₂O/HOAc (50:49:1, v/v/v)) was applied to the rear end ofthe triangle paper. Due to the capillary effect, the solvent movedforward and dissolved the chemicals in the blood spot. Finally,electrospray occurred when the solvent reached the tip of the paper. Todemonstrate the effectiveness of the “blood spot” method mentionedabove, the whole blood was added on the paper for electrospray directly.MS/MS spectrum showed that heroin was not detected from 10 μl of wholeblood sample, even when the concentration was as high as 20 ppm (FIG.7B).

FIG. 8A shows an MS/MS spectrum that shows that heroin can be detectedfrom raw urine sample by a “spot” method. 0.4 μl of raw urine samplecontaining 100 ppb heroin was applied on the center of the trianglepaper to form a 1 mm² urine spot. After the spot was dry, 10 μl ofsolvent (MeOH/H₂O/HOAc (50:49:1, v/v/v)) was applied to the rear end ofthe triangle paper. Due to the capillary effect, the solvent movedforward and dissolved the chemicals in the blood spot. Finally,electrospray occurred when the solvent reached the tip of the paper. Todemonstrate the effectiveness of the “spot” method mentioned above, theraw urine was added on the paper for electrospray directly. MS/MSspectrum showed heroin was not detected from 10 μl of raw urine samplewhen concentration was 100 ppb (FIG. 8B).

FIG. 9A is an MS spectrum showing that caffeine was detected from a coladrink without sample preparation. FIG. 9B is an MS spectrum showing thatcaffeine was detected from coffee powder. A paper triangle was used tocollect the coffee powder from a coffee bag by swabbing the surface.

FIGS. 22A-B show the spectra of COCA-COLA (cola drink), analyzed inpositive mode and negative mode, respectively. The peak of protonatedcaffeine, m/z 195, identified in MS/MS spectrum, was dominated in themass spectrum in positive mode due to the high concentration of caffeine(100 ug/mL) in this drink (FIG. 22C). Two high concentrated compounds,potassium benzoate and acesulfame potassium were identified in the MS/MSspectrum in negative mode (FIGS. 22D-E).

FIG. 22F shows spectra of caffeine in urine from a person who had drunkCOCA-COLA (cola drink) two hours before the urine collection. Urinetypically contains urea in very high concentration, which is also easilyionized. Therefore, protonated urea [m/z, 61] and urea dimmer [m/z, 121]dominated the MS spectrum. However, the protonated caffeine wasidentified in the MS/MS spectrum, which showed good signal to noiseratio in the urine sample.

FIGS. 10A-B show MS spectra of urine taken for analysis without samplepreparation. FIG. 10A is a mass spectra of caffeine that was detected inurine from a person who had consumed coffee. FIG. 10B is a mass spectrashowing that caffeine was not detected in urine from a person who hadnot consumed any coffee.

FIG. 22G shows the MS spectrum of heroin (m/z, 370) collected as aswabbed sample. A 5 uL solution containing 50 ng heroin was spotted on a1 cm² area of a desktop. The paper triangle was wetted and used to swabthe surface of the desktop. The paper triangle was then connected to thehigh voltage source for mass detection. This data shows that probes ofthe invention can have dual roles of ionization source as well as asampling device for mass detection. Trace sample on solid surface couldbe simply collected by swabbing the surface using probes of theinvention. Dust and other interferences were also collected on the papertriangle, but the heroin could be directly detected from this complexmatrix.

Example 8 Plant Tissue Direct Analysis by ESI without Extraction

FIGS. 12A-D shows direct MS spectra of plant tissues using slicedtissues of four kinds of plants. (FIG. 12A) Onion, (FIG. 12B) Springonion, and two different leaves (FIG. 12C) and (FIG. 12D).

FIGS. 13A-B shows an MS/MS spectra of Vitamin C analysis (FIG. 13A)direct analysis of onion without sample preparation, (FIG. 13B) usingstandard solution.

Example 9 Whole Blood and Other Biofluids

Body fluids, such as plasma, lymph, tears, saliva, and urine, arecomplex mixtures containing molecules with a wide range of molecularweights, polarities, chemical properties, and concentrations. Monitoringparticular chemical components of body fluids is important in a numberof different areas, including clinical diagnosis, drug development,forensic toxicology, drugs of abuse detection, and therapeutic drugmonitoring. Tests of blood, including the derived fluids plasma andserum, as well as on urine are particularly important in clinicalmonitoring.

A wide variety of chemicals from blood are routinely monitored in aclinical setting. Common examples include a basic metabolic panelmeasuring electrolytes like sodium and potassium along with urea,glucose, and creatine and a lipid panel for identifying individuals atrisk for cardiovascular disease that includes measurements of totalcholesterol, high density lipoprotein (HDL), low density lipoprotein(LDL), and triglycerides. Most laboratory tests for chemicals in bloodare actually carried out on serum, which is the liquid component ofblood separated from blood cells using centrifugation. This step isnecessary because many medical diagnostic tests rely on colorimetricassays and therefore require optically clear fluids. Aftercentrifugation, detection of the molecule of interest is carried in anumber of ways, most commonly by an immunoassay, such as anenzyme-linked immunosorbent assay (ELISA) or radioimmunoas say (RIA), oran enzyme assay in which the oxidation of the molecule of interest by aselective enzyme is coupled to a reaction with a color change, such asthe tests for cholesterol (oxidation by cholesterol oxidase) or glucose(oxidation by glucose oxidase).

There is considerable interest in the pharmaceutical sciences in thestorage and transportation of samples of whole blood as dried bloodspots on paper (N. Spooner et al. Anal Chem., 2009, 81, 1557). Mosttests for chemicals found in blood are carried out on a liquid sample,typically serum or plasma isolated from the liquid whole blood. Therequired storage, transportation, and handling of liquid blood or bloodcomponents present some challenges. While blood in liquid form isessential for some tests, others can be performed on blood or other bodyfluids that have been spotted onto a surface (typically paper) andallowed to dry.

Probes and methods of the invention can analyze whole blood without theneed for any sample preparation. The sample was prepared as follows. 0.4uL blood was directly applied on the center of paper triangle and leftto dry for about 1 min. to form a dried blood spot (FIG. 23A). 10 uLmethanol/water (1:1, v/v) was applied near the rear end of the papertriangle. Driven by capillary action, the solution traveled across thepaper wetting it throughout its depth. As the solution interacted withthe dried blood spot, the analytes from the blood entered the solutionand were transported to the tip of the probe for ionization (FIG. 23A).The process of blood sample analysis was accomplished in about 2 min.

Different drugs were spiked into whole blood and the blood was appliedto probes of the invention as described above. Detection of differentdrugs is described below.

Imatinib (GLEEVEC), a 2-phenylaminopyrimidine derivative, approved bythe FDA for treatment of chronic myelogenous leukemia, is efficaciousover a rather narrow range of concentrations. Whole human blood, spikedwith imatinib at concentrations including the therapeutic range, wasdeposited on a small paper triangle for analysis (FIG. 14A). The tandemmass spectrum (MS/MS, FIG. 14B) of protonated imatinib, m/z 494, showeda single characteristic fragment ion. Quantitation of imatinib in wholeblood was achieved using this signal and that for a known concentrationof imatinib-d8 added as internal standard. The relative response waslinear across a wide range of concentrations, including the entiretherapeutic range (FIG. 14C).

Atenolol, a β-blocker drug used in cardiovascular diseases, was testedusing the dried blood spot method to evaluate paper spray for wholeblood analysis. Atenolol was directly spiked into whole blood at desiredconcentrations and the blood sample was used as described above forpaper spray. The protonated atenolol of 400 pg (1 ug/mL atenolol in 0.4uL whole blood) in dried blood spot was shown in mass spectra, and theMS/MS spectra indicated that even 20 pg of atenolol (50 ug/mL atenololin 0.4 uL whole blood) could be identified in the dried blood spot (FIG.23B).

FIG. 23C is a mass spectra of heroin in whole blood. Data herein showthat 200 pg heroin in dried blood spot could be detected using tandemmass.

It was also observed that the paper medium served a secondary role as afilter, retaining blood cells. Significantly, samples were analyzeddirectly on the storage medium rather than requiring transfer from thepaper before analysis. All experiments were done in the open labenvironment. Two additional features indicated that the methodology hadthe potential to contribute to increasing the use of mass spectrometryin primary care facilities: blood samples for analysis were drawn bymeans of a pinprick rather than a canula; and the experiment was readilyperformed using a handheld mass spectrometer (FIG. 18 and Example 10below).

Example 10 Handheld Mass Spectrometer

Systems and methods of the invention were compatible with a handheldmass spectrometer. Paper spray was performed using a handheld massspectrometer (Mini 10, custom made at Purdue University). Analysis ofwhole blood spiked with 10 μg/mL atenolol. Methanol/water (1:1; 10 μL)was applied to the paper after the blood (0.4 uL) had dried (˜1 min) togenerate spray for mass detection (FIG. 18). The inset shows thatatenolol could readily be identified in whole blood using tandem massspectrum even when the atenolol amount is as low as 4 ng.

Example 11 Angiotensin I

FIG. 15 is a paper spray mass spectrum of angiotensin I solution(Asp-Arg-Val-Tyr-Ile-His-Pro-Phe-His-Leu (SEQ ID NO: 1), 10 μL, 8 μg/mLin methanol/water, 1:1, v/v) on chromatography paper (spray voltage, 4.5kV). The inset shows an expanded view over the mass range 630-700. Theprotonated ([M+2H]²⁺) and sodium-adduct ions ([M+H+Na]²⁺, [M+2Na]²⁺) arethe major ionic species.

Example 12 Agrochemicals on Fruit

Sample collection by paper wiping followed by analysis using probes ofthe invention was used for fast analysis of agrochemicals on fruit.Chromatography paper (3×3 cm) wetted with methanol was used to wipe a 10cm² area on the peel of a lemon purchased from a grocery store. Afterthe methanol had dried, a triangle was cut from the center of the paperand used for paper spray by applying 10 μL methanol/water solution. Thespectra recorded (FIGS. 34A-B) show that a fungicide originally on thelemon peel, thiabendazole (m/z 202 for protonated molecular ion and m/z224 for sodium adduct ion), had been collected onto the paper and couldbe identified easily with MS and confirmed using MS/MS analysis. Anotherfungicide imazalil (m/z 297) was also observed to be present.

Example 13 Tumor Sample

Systems and methods of the invention were used to analyze human prostatetumor tissue and normal tissue. Tumor and adjacent normal tissuesections were 15 μm thick and fixed onto a glass slide for an imagingstudy using desorption electrospray ionization (DESI). A metal needlewas used to remove a 1 mm²×15 μm volume of tissue from the glass slidefrom the tumor region and then from the normal region and place themonto the surface of the paper triangle for paper spray analysis.

A droplet of methanol/water (1:1 v:v; 10 μl) was added to the paper assolvent and then 4.5 kV positive DC voltage applied to produce thespray. Phospholipids such as phosphatidylcholine (PC) and sphingomyelin(SM) were identified in the spectrum (FIGS. 17A-B). The peak of[PC(34:1)+K]⁺ at m/z 798 was significantly higher in tumor tissue andpeaks [SM(34:1)+Na]⁺ at m/z 725, [SM(36:0)+Na]⁺ at m/z 756, and[SM(36:4)+Na]⁺ at m/z 804 were significantly lower compared with normaltissue.

Example 14 Therapeutic Drug Monitoring

The administration of a drug depends on managing the appropriate dosingguidelines for achievement of a safe and effective outcome. Thisguideline is established during clinical trials where thepharmacokinetics (PK) and pharmacodynamics (PD) of the drug are studied.Clinical trials use PK-PD studies to establish a standard dose, whichmay be fixed or adjusted according formulas using variables like bodymass, body surface area, etc. However, the drug exposure, i.e. theamount of drug circulating over time, is influenced by a number offactors that vary from patient to patient. For example, an individuals'metabolic rate, the type and level of plasma proteins, and pre-existingconditions such as renal and/or hepatic impairment all play a role inaffecting the exposure of the drug in vivo. Further, administration of adrug in combination with other medications may also affect exposure. Asa result, it is often difficult to predict and prescribe an optimumregimen of drug administration.

Over- or underexposure to a drug can lead to toxic effects or decreasedefficacy, respectively. To address these concerns, therapeutic drugmonitoring (TDM) can be employed. TDM is the measurement of active druglevels in the body followed by adjustment of drug dosing or schedules toincrease efficacy and/or decrease toxicity. TDM is indicated when thevariability in the pharmacokinetics of a drug is large relative to thetherapeutic window, and there is an established relationship betweendrug exposure and efficacy and/or toxicity. Another requirement for TDMis that a sufficiently precise and accurate assay for the activeingredient must be available. Immunoassays and liquid chromatographymass spectrometry (LC-MS) are commonly used methods for TDM. Incomparison with immunoassay, LC-MS has advantages which include wideapplicability, high sensitivity, good quantitation, high specificity andhigh throughput. Probes of the invention may be coupled with standardmass spectrometers for providing point-of-care therapeutic drugmonitoring.

The drug Imatinib (GLEEVEC in USA and GLIVEC in Europe/Australia, forthe treatment of chronic myelogenous leu-kemia) in a dried blood spotwas analyzed using paper spray and a lab-scale LTQ mass spectrometer.Quantitation of Imatinib in whole blood was achieved using the MS/MSspectra with a known concentration of Imatinib-d8 being used as theinternal standard (FIG. 14C). The relative response was linear across awide range of concentrations, including the entire therapeutic range(FIG. 14C).

Example 15 High-Throughout Detection

Multiple-tip devices were fabricated and applied for high throughputanalysis (FIG. 28A). The multiple-tip device was a set of papertriangles all connected to a single copper strip (FIG. 28A). Anelectrode was connected to the copper strip. Multiple samples were puton a single paper substrate and analyzed in series using themultiple-tip probe (FIGS. 28B-C). Each tip was pre-loaded with 0.2 uLmethanol/water containing 100 ppm sample (cocaine or caffeine) anddried. Then the whole multiple-tip device was moved on a moving stagefrom left to right with constant velocity and 7 uL methanol/water wasapplied from the back part for each tip during movement.

To prevent the contaminant during spray, blanks were inserted betweentwo sample tips. FIG. 28C shows the signal intensity for the wholescanning. From total intensity, six tips gave six individual high signalpeaks. For cocaine, peaks only appeared when tip 2 and tip 6 werescanned. For caffeine, the highest peak came from tip 4, which wasconsistent with the sample loading sequence.

Example 16 Tissue Analysis

Direct analysis of chemicals in animal tissue using probes of theinvention was performed as shown in FIG. 29A. A small sections of tissuewere removed and placed on a paper triangle. Methanol/water (1:1 v:v; 10μl) was added to the paper as solvent and then 4.5 kV positive DCvoltage was applied to produce the spray for MS analysis. Protonatedhormone ions were observed for porcine adrenal gland tissue (1 mm³, FIG.29B). FIG. 16 is a mass spectrum showing direct analysis of hormones inanimal tissue by paper spray. A small piece of pig adrenal gland tissue(1 mm×1 mm×1 mm) was placed onto the paper surface, MeOH/water (1:1 v:v;10 μl) was added and a voltage applied to the paper to produce a spray.The hormones epinephrine and norepinephrine were identified in thespectrum; at high mass the spectrum was dominated by phospholipidsignals.

Lipid profiles were obtained for human prostate tissues (1 mm²×15 μm,FIGS. 29C-D) removed from the tumor and adjacent normal regions.Phospholipids such as phosphatidylcholine (PC) and sphingomyelin (SM)were identified in the spectra. The peak of [PC(34:1)+K]⁺ at m/z 798 wassignificantly more intense in tumor tissue (FIG. 29C) and peaks[SM(34:1)+Na]⁺ at m/z 725, [SM(36:0)+Na]⁺ at m/z 756, and [SM(36:4)+Na]⁺at m/z 804 were significantly lower compared with normal tissue (FIG.29D).

Example 17 On-Line Derivatization

For analysis of target analytes which have relatively low ionizationefficiencies and relatively low concentrations in mixtures,derivatization is often necessary to provide adequate sensitivity.On-line derivatization can be implemented by adding reagents into thespray solution, such as methanol/water solutions containing reagentsappropriate for targeted analytes. If the reagents to be used are stableon paper, they can also be added onto the porous materal when the probesare fabricated.

As a demonstration, 5 μL methanol containing 500 ng betaine aldehydechloride was added onto a paper triangle and allowed to dry to fabricatea sample substrate preloaded with a derivatization reagent for theanalysis of cholesterol in serum. On-line charge labeling with betainealdehyde (BA) through its reaction with hydroxyl groups has beendemonstrated previously to be very effective for the identification ofcholesterol in tissue (Wu et al., Anal Chem. 2009, 81:7618-7624). Whenthe paper triangle was used for analysis, 2 μL human serum was spottedonto the paper to form a dried spot and then analyzed by using paperspray ionization. A 10 μL ACN/CHCl₃ (1:1 v:v) solution, instead ofmethanol/water, was used for paper spray to avoid reaction between thebetaine aldehyde and methanol.

The comparison between analysis using a blank and a reagent-preloadedpaper triangle is shown in FIGS. 30A-B. Without the derivatizationreagent, cholesterol-related peaks, such as protonated ion [Chol+H]⁺(m/z 387), water loss [Chol+H−H₂O]⁺ (m/z 369), and sodium adduction[Chol+Na]⁺ (m/z 409), were not observed (FIG. 30A). With thederivatization reagent, the ion [Chol+BA]⁺ was observed at m/z 488.6(FIG. 30B). MS/MS analysis was performed for this ion and acharacteristic fragment ion m/z 369 was observed (FIG. 30C).

Example 18 Peptide Pre-Concentration Using Modified Paper SpraySubstrate

Pre-concentration of chemicals on the paper surface using photoresisttreatment. Chromatography paper was rendered hydrophobic by treatmentwith SU-8 photoresist as described previously (Martinez et al., AngewChem. Int. Ed., 2007, 46:1318-1320). Then 5 μl bradykinin 2-9 solution(100 ppm in pure H₂O) was applied on the paper surface. When thesolution was dry, the paper was put into water and washed for 10 s.After washing, the paper triangle was held in front of the MS inlet, 10μl pure MeOH was applied as solvent and the voltage was set at 4.5 kVfor paper spray. The same experiment was done with untreated papersubstrate for comparison.

FIG. 31A shows the tandem MS spectrum of bradykinin 2-9 from paper withphotoresist treatment. The intensity of the most intense fragment ion404 is 5.66E3. FIG. 31B shows the tandem MS spectrum of bradykinin 2-9from normal chromatography paper without photoresist treatment. Theintensity of the most intense fragment ion 404 is only 1.41E1. Thesedata show that the binding affinity between photoresist-treatedchromatography paper and peptide is much higher than that between normalchromatography paper and peptide, thus more peptide can be kept on thepaper surface after washing by water. When pure methanol is applied,these retained peptides will be desorbed and detected by MS. This methodcan be used to pre-concentrate hydrophobic chemicals on the papersurface, and other hydrophilic materials (e.g. salts) can also beremoved from the paper surface.

Example 19 Inverted Polarities

The polarity of the voltage applied to the probe need not match thatused in the mass analyzer. In particular, it is possible to operate theprobes of the invention with a negative potential but to record the massspectrum of the resulting positively changed ions. In negative ion mode,a large current of electrons (or solvated electrons) is produced inpaper spray. These electrons, if of suitable energy, can be captured bymolecules with appropriate electron affinities to generate radicalanions.

Alternatively, these electrons might be responsible for electronionization of the analyte to generate the radical cation oralternatively ESI might involve a solvent molecule which might thenundergo charge exchange with the analyte to again generate the radicalcation. If this process occurs with sufficient energy, characteristicfragment ions might be produced provided the radical cation is notcollisionally deactivated before fragmentation can occur.

An experiment was done on a benchtop LTP using toluene vapor, with aprobe of the invention conducted at −4.5 kV with methanol:water assolvent applied to the paper. The spectrum shown in FIG. 32 wasrecorded. One notes that ion/molecule reactions to give the protonatedmolecule, m/z 93 occur as expected at atmospheric pressure. One alsonotes however, the presence of the radical cation, m/z 92 and itscharacteristic fragments at m/z 91 and 65.

An interesting note is that the “EI” fragment ions were most easilyproduced when the source of toluene vapor was placed close to the MSinlet; i.e., in the cathodic region of the discharge between the papertip and MS inlet. This suggests that direct electron ionization byenergetic electrons in the “fall” region might be at least partlyresponsible for this behavior.

Example 20 Cartridge for Blood Analysis

FIG. 33A shows an exemplary case for spotting blood onto porous materialthat will be used for mass spectral analysis. The cartridge can have avial with a volume at the center and vials for overflows. A plug, suchas a soluble membrane containing a set amount of internal standardchemical, is used to block the bottom of the vial for volume control. Adrop of blood is placed in the vial (FIG. 33B). The volume of the bloodin the vial is controlled by flowing the extra blood into the overflowvials (FIG. 33B). The blood in the vial is subsequently dissolved in themembrane at the bottom, mixing the internal standard chemical into theblood (FIG. 33B). Upon dissolution of the plug, blood flows to the papersubstrate, and eventually forms a dried blood spot having a controlledamount of sample and internal standard (FIG. 33B).

Example 21 Microorganism Analysis and Identification

In certain aspects, probes of the invention can be used to analyze oneor more microorganisms (e.g., bacteria, viruses, protozoans (alsospelled protozoon), or fungi) in a sample. An exemplary method involvescontacting a sample including a microorganism to a porous material, inwhich the porous material is kept separate from a flow of solvent. Themethod further involves applying high voltage to the porous material togenerate ions of the microorganism that are expelled from the porousmaterial, and analyzing the expelled ions, thereby analyzing themicroorganism. Methods of the invention may also involve applying asolvent to the porous material. Any mass spectrometer known in the artmay be used, and in certain embodiments, the mass spectrometer is aminiature mass spectrometer, such as that described for example in Gaoet al. (Anal. Chem., 80:7198-7205, 2008) and Hou et al. (Anal. Chem.,83:1857-1861, 2011), the content of each of which is incorporated hereinby reference herein in its entirety.

The sample may be any type of sample and may be in any form, forexample, a solid, a liquid, or a gas. In certain embodiments, the sampleis a human tissue or body fluid. The sample may be an in vivo sample oran extracted sample. In certain embodiments, the methods of theinvention are sensitive enough to analyze and identify microorganismswithout first culturing the microorganism. In some embodiments, themicroorganism is cultured prior to analysis, however, methods of theinvention allow for decreased culture time over that used in standardprocedures.

FIG. 37 shows an exemplary method of collecting microorganisms ontoprobes of the invention when the sample is a gas/aerosol. The figureshows a probe of the invention housed within a cartridge. Suchcartridges are described for example in PCT/US12/40513, the content ofwhich is incorporated by reference herein in its entirety. Air is flowedthrough the cartridge, which causes, under appropriate fluid flowconditions, any microorganisms in the air to flow onto the substratehoused in the cartridge. Once collected, voltage and a discrete amountof solvent is applied to the probe, and ions of the microorganism aregenerated and expelled into a mass spectrometer for analysis.

FIG. 38 shows an exemplary method of collecting microorganisms ontoprobes of the invention when the sample is a liquid. As shown in thefigure, a porous substrate is loaded into the funnel. A liquid samplecontaining bacteria poured into the funnel and vacuum is used to pullthe sample through the porous substrate and into a collection vessel. Asthe sample passes through the porous substrate, the microorganism isretained on the porous substrate. Once collected, voltage and a discreteamount of solvent is applied to the probe, and ions of the microorganismare generated and expelled into a mass spectrometer for analysis. FIGS.39A-B show mass spectra of E. coli generated using the set-up asdescribed in FIG. 38. FIG. 39A is negative ion mode and FIG. 39B ispositive ion mode.

If the sample is a solid, as in the case of a microorganism grown onagar in a petri dish for example, the porous substrate can be contactedto the solid and swapped or moved across the surface such thatmicroorganisms in the sample are retained by the porous substrate. Thereare other methods for collecting microorganisms from solid samples. Forexample, for colonies on petri dishes, a sterile inoculation loop isused to transfer one or more colonies to the porous substrate. Oncecollected, voltage and a discrete amount of solvent is applied to theprobe, and ions of the microorganism are generated and expelled into amass spectrometer for analysis.

Regardless of the collection method, voltage, and optionally a discreteamount of solvent, is applied to the probe, and ions of themicroorganism are generated and expelled into a mass spectrometer foranalysis. FIGS. 40-42 shows mass spectra of microorganisms analyzedusing probes and methods of the invention.

Aspects of the invention also provide methods of identifying anorganism, e.g., a microorganism. The methods include obtaining a massspectrum of an organism using porous substrate probes of the inventionand correlating/comparing the mass spectra with a database that includesmass spectra of known organisms (FIGS. 43-45A-F). With use of methods ofthe invention, the organism can be identified and classified not just ata genus and species level, but also at a sub-species (strain), asub-strain, and/or an isolate level. The featured methods offer fast,accurate, and detailed information for identifying organisms. Themethods can be used in a clinical setting, e.g., a human or veterinarysetting; or in an environmental, industrial or forensic/public safetysetting (e.g., clinical or industrial microbiology, food safety testing,ground water testing, air testing, contamination testing, and the like).In essence, the invention is useful in any setting in which thedetection and/or identification of a microorganism is necessary ordesirable.

A database for use in the invention can include a similarity cluster.The database can be used to establish, through linear discriminantanalysis and related methods, a quantitative measure of the similarityof any two spectra, a similarity index. The database can include a massspectrum from at least one member of the Clade of the organism. Thedatabase can include a mass spectrum from at least one subspecies of theorganism. The database can include a mass spectrum from a genus, aspecies, a strain, a sub-strain, or an isolate of the organism. Thedatabase can include a mass spectrum with motifs common to a genus, aspecies, a strain, a sub-strain, or an isolate of the organism.

The database(s) used with the methods described herein includes massspectra associated with known organisms (FIGS. 45A-F). The mass spectraare typically annotated to show if they were acquired in positive ornegative mode. The database(s) can contain information for a largenumber of isolates, e.g., about 200, about 300, about 400, about 500,about 600, about 700, about 800, about 900, about 1,000, about 1,500,about 2,000, about 3,000, about 5,000, about 10,000 or more isolates. Inaddition, the mass spectra of the database contain annotated information(a similarity index or cluster, see FIGS. 43-44) regarding motifs commonto genus, species, sub-species (strain), sub-strain, and/or isolates forvarious organisms. The large number of the isolates and the informationregarding specific motifs allows for accurate and rapid identificationof an organism. The data in FIG. 43 show that there is separation offungi and bacteria, a separation between gram negative and gram positivebacteria, and a separation of gram negative species.

To generate similarity clusters, each mass spectrum is aligned againstevery other mass spectrum. From these alignments, a pair-wise alignmentanalysis is performed to determine “percent dissimilarity” between themembers of the pair (FIG. 44). Briefly, this clustering method works byinitially placing each entry in its own cluster, then iterativelyjoining the two nearest clusters, where the distance between twoclusters is the smallest dissimilarity between a point in one clusterand a point in the other cluster.

Various organisms, e.g., viruses, and various microorganisms, e.g.,bacteria, protists, and fungi, can be identified with the methodsfeatured herein. The sample containing the organism to be identified canbe a human sample, e.g., a tissue sample, e.g., epithelial (e.g., skin),connective (e.g., blood and bone), muscle, and nervous tissue, or asecretion sample, e.g., saliva, urine, tears, and feces sample. Thesample can also be a non-human sample, e.g., a horse, camel, llama, cow,sheep, goat, pig, dog, cat, weasel, rodent, bird, reptile, and insectsample. The sample can also be from a plant, water source, food, air,soil, plants, or other environmental or industrial sources.

The methods described herein include correlating the mass spectrum fromthe unknown organism with a database that includes mass spectra of knownorganisms. The methods involve comparing each of the mass spectra fromthe unknown organism from a sample against each of the entries in thedatabase, and then combining match probabilities across differentspectra to create an overall match probability (FIGS. 45A-F).

1-47. (canceled)
 48. A microorganism identification system, the systemcomprising: a capture module, the module configured to capture anunknown microorganism from a sample and generate ions of the unknownmicroorganism, wherein the capture module comprises a porous substrateoperably coupled to a high voltage source; a mass analyzer operablycoupled to the capture module to receive the generated ions of theunknown microorganism; and a central processing unit (CPU) and storagecoupled to the CPU for storing instructions that when executed by theCPU cause the CPU to: accept as input from the mass analyzer, at leastone mass spectrum of the unknown microorganism; compare the at least onemass spectrum of the unknown microorganism to a database comprising massspectra of known organisms; and output an identification of the unknownorganism based on results of the comparison.
 49. The system according toclaim 48, wherein the sample is a liquid sample.
 50. The systemaccording to claim 48, wherein the sample is a gas sample.
 51. Thesystem according to claim 48, wherein the sample is a solid sample. 52.The system according to claim 48, wherein the porous substrate isdiscrete from a flow of solvent.
 53. The system according to claim 48,wherein the porous substrate tapers to at least one distal tip.
 54. Thesystem according to claim 48, further comprising a solvent applied tothe porous substrate.
 55. The system according to claim 54, wherein thesolvent comprises an internal standard.
 56. The system according toclaim 48, wherein the microorganism is a non-natural microorganism. 57.The system according to claim 48, wherein the mass analyzer is for amass spectrometer or a handheld mass spectrometer.
 58. The systemaccording to claim 57, wherein the mass analyzer is selected from thegroup consisting of: a quadrupole ion trap, a rectalinear ion trap, acylindrical ion trap, a ion cyclotron resonance trap, an orbitrap, atime of flight, a Fourier Transform ion cyclotron resonance, andsectors.
 59. The system according to claim 48, wherein the databasecomprises a similarity cluster.
 60. The system of claim 48, wherein thedatabase comprises a mass spectrum from at least one member of a cladeof organisms.
 61. The system of claim 48, wherein the database comprisesa mass spectrum from at least one subspecies of organisms.
 62. Thesystem of claim 48, wherein the database comprises a mass spectrum froma genus, a species, a strain, a sub-strain, or an isolate of organisms.63. The system of claim 48, wherein the database comprises a massspectrum comprising motifs common to a genus, a species, a strain, asub-strain, or an isolate of organisms.
 64. The system according toclaim 48, wherein the mass spectra in the database are annotated to showif they were acquired in positive or negative mode.
 65. The systemaccording to claim 48, wherein the microorganism is a bacterium.
 66. Thesystem according to claim 48, wherein the microorganism is a virus. 67.The system according to claim 48, wherein the microorganism is a fungus.